Stable Expression by Lentiviral Transduction of Cells

Gödecke, Natascha; Hauser, Hansjörg; Wirth, Dagmar

doi:10.1007/978-1-4939-8730-6_4

Cited by 8 publications

(8 citation statements)

References 8 publications

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“…The recombinant CHO cells were obtained using LVs. One advantage of LVs, is the possibility to in uence the transgene copy number and/or the percentage of infected cells by adjusting the ratio of virus/cell (Gödecke et al, 2018;Oberbek et al, 2011). In this way, an increase of MOI values could mean higher copy number of DNA of interest inside the genome of host cells and, therefore, higher levels of expression of recombinant CHO cells.…”

Section: Discussionmentioning

confidence: 99%

“…On another hand, these cells can be easily transduced using lentiviral vectors (LVs) (Gaillet et al, 2010;Oberbek et al, 2011). This transfection method takes advantage of LV mechanisms for stable integration within the chromosome of the cells, speci cally into transcriptionally open chromatin, which makes them excellent tools to obtain cell lines with high expression levels of recombinant proteins (Gödecke et al, 2018;Mursi and Masuda, 2018). In addition, the approach of LVs as gene transfer method is suitable because it allows the adjustment of the ratio of virus/cell (multiplicity of infection, MOI) and, therefore, a cha;nge on transgene copy number and/or the percentage of infected cells (Oberbek et al, 2011;Gödecke et al, 2018).…”

mentioning

confidence: 99%

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Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Lao-González

Soler

Hernandez

et al. 2020

Preprint

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Abstract The high prices of biopharmaceutics or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceutics. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines in a short period of time with higher levels of expression. Here, we obtain trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein free media and suspension culture and lentiviral vectors. The results shown that early screening strategy allowed to obtain recombinant CHO cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular HC polypeptides by flow cytometry was a useful tool to characterize the homogeneity of cell population and our results suggest that might be used to predict the expression levels of monoclonal antibodies in early stages of cell line development process. Furthermore, using T-flask approach the assessment of the expression levels was studied in a setting more similar to that of a final production process. Finally, trastuzumab-expressing CHO-K1 clones were generated, characterized by batch experiments and preliminary results related to HER2-recognition capacity were successful. Further improvements in up-stream and down-stream steps related to this strategy will improve specific productivities and volumetric productivities of these clones.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Lao-González

Soler

Hernandez

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…The recombinant CHO-K1 cells were obtained using LVs. One advantage of LVs, is the possibility to in uence the transgene copy number and/or the percentage of infected cells by adjusting the virus to cell ratio (Gödecke et al 2018;Oberbek et al 2011). In this way, increased MOI values could mean higher copy number of the DNA of interest inside the genome of host cells; and therefore, higher levels of target protein expression.…”

Section: Discussionmentioning

confidence: 99%

“…This transfection method takes advantage of LV mechanisms for stable integration within the chromosome of the cells, speci cally into transcriptionally active regions. This feature makes them excellent tools for obtaining cell lines with high expression levels of target recombinant proteins (Gödecke et al 2018;Mursi and Masuda 2018). LVs as a gene transfer method provides additional opportunities for optimization as it allows the adjustment of the virus to cell ratio (multiplicity of infection, MOI) to modulate transgene copy number and/or the percentage of infected cells (Gödecke et al 2018;Oberbek et al 2011).…”

mentioning

confidence: 99%

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Lao-González

Soler

Hernandez

et al. 2020

Preprint

View full text Add to dashboard Cite

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

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“…This transfection method takes advantage of LV mechanisms for stable integration within the chromosome of the cells, specifically into transcriptionally active regions. This feature makes them excellent tools for obtaining cell lines with high expression levels of target recombinant proteins (Gödecke et al 2018 ; Mursi and Masuda 2018 ). LVs as a gene transfer method provides additional opportunities for optimization as it allows the adjustment of the virus to cell ratio (multiplicity of infection, MOI) to modulate transgene copy number and/or the percentage of infected cells (Gödecke et al 2018 ; Oberbek et al 2011 ).…”

Section: Introductionmentioning

confidence: 99%

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

et al. 2021

View full text Add to dashboard Cite

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

show abstract

Stable Expression by Lentiviral Transduction of Cells

Cited by 8 publications

References 8 publications

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

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